Follicular development and differentiation, key to female reproduction, are sequential events that are tightly regulated by balanced cell proliferation, survival and cell death. Previously we reported the prohibitins, PHB1 and REA, as important mediators of the effects of gonadotropins on granulosa cell (GC) differentiation and apoptosis. Consistent with our long term goal of understanding their functions in folliculogenesis, new preliminary data here underscores their major role in GC growth and differentiation. We show that there is a clear association between PHB1 expression and follicular development in vivo. Furthermore, PHB1 expression is differentially regulated in response to FSH plus activin (A) in proliferation (FSH+A??PHB1) vs. FSH plus testosterone (T) in differentiation (FSH+T??PHB1) in vitro. Forced expression of PHB1 impairs cell cycle progression and down regulation results in a proliferative phenotype in GCs. PHB1 is phosphorylated on Y249 in a MEK1-dependent fashion while PHB1 is required for ERK1/2 activation defining a new regulatory paradigm in response to FSH+T in primary GCs. These observations support our central hypothesis that prohibitins are critical intracellular mediators of FSH stimulation of GCs, affecting follicular development, and the derived prediction that FSH+T-dependent MEK1-mediated phosphorylation of PHB1 is essential for its function in GCs differentiation. In this proposal we will address these two essential premises using a systematic experimental design that combines gain- and loss-of-function, mutagenesis, genetic approaches, biochemical fractionation and immunostaining in primary GCs in vitro in response to FSH- dependent physiological synergistic stimuli with either activin or testosterone. A GC specific conditional knock- out PHB1 animal model will be used to circumvent the embryonic lethality identified for PHB1 and address the in vivo role of PHB1. Specifically, we propose: Aim 1: To test the hypothesis that PHB1 and REA levels determine the differential effects of FSH in proliferation and differentiation of GCs in vitro; Aim 2: To test the hypothesis that PHB1 is obligatory for folliculogenesis in vivo; and Aim 3: To test the hypothesis that PHB1 functions as a scaffold and substrate to coordinate FSH+T signaling in GCs differentiation. Unraveling the mechanisms underlying the growth and development of a competent follicle with the capacity t release a fertilizable oocyte is a major goal of basic reproductive biology research. PUBLIC HEALTH RELEVANCE: Understanding of the molecular mechanism of prohibitins in GCs growth and differentiation in the normal physiological development of the follicle as well as its role in the pathogenesis of ovarian dysfunction could have profound implications in the clinical setting in the field of fertility (contraception or infertility), and lead to development of novel molecular diagnostic and therapeutic tools (population based screenings, ovarian cancer).